The application has the goal to develop an improved vaccine to rabies virus that provides protection upon a single dose given orally or intranasally against a severe form of inhalation challenge such as with weaponized rabies virus. We have developed an El-deleted adenoviral recombinant derived from a chimpanzee isolate. This adenoviruses (Ad) of the C68 serotype (AdC68) does not circulate in the human population and virus neutralizing antibodies (VNAs) to common human serotypes of Ad fail to cross-react with AdC68 virus. Vaccines based on AdC68 virus, such as the one expressing the rabies virus glycoprotein (AdC68rab.gp) are thus not impaired by natural exposure of a human population to most common human serotypes of Ad. In aim 1, the biodistribution of the AdC68rab.gp vaccine upon oral and intranasal application into mice will be tested. It will be established if oral or intranasal immunization with the AdC68rab.gp vaccine protects against inhalation rabies and which immune effect mechanisms (serum or mucosal VNAs or frequencies of rabies virus specific B cells in nasal associated lymphoid tissues, spleens or lungs) correlate with protection. The longevity of vaccine-induced protection will be determined. Efficacy of the mucosal vaccine will be assessed in immunologically challenged animals such as infant or aged mice and mice lacking CD4+ T cells. In aim 2, the AdC68rab.gp vaccine will be tested in a non-human primate rabies virus challenge model.